PROJECT SUMMARY The cost of new therapy discovery has continued to increase and is estimated to be near $2.6 billion per compound. Technological advances allowing for more effective screening could have an immense clinical benefit and reduce the cost of drug discovery. Gains in efficiency for compound screening could come through direct introduction of drug candidates into cells, development of better cell models, and improved analysis methods such as high content live cell imaging- all depending on reliable and scalable delivery of materials of interest into cells. Existing delivery technologies based on nanoparticle, detergent, and liposome methods struggle to accommodate the chemical diversity of materials and often lead to heightened toxicity and off-target effects, limiting their utility. There is a pressing need to develop better delivery methods to deliver diverse material types into various cell types at a scale relevant to drug discovery. In SBIR Phase I, SQZ Biotech?s CellSqueeze that opens up transient pores on cell membrane allowing materials to diffuse in by gently squeezing cells has demonstrated tremendous potential to address the current delivery issues in drug discovery with improvement on device design and throughput successfully achieved and >70% delivery to cells of materials including small molecules, peptides, antibodies and proprietary materials successfully demonstrated. When compared to alternative technologies, CellSqueeze has also demonstrated superior delivery and functional performance, which sets it apart from competitors. In this Phase II SBIR application, SQZ proposes to develop a high capacity (AIM 1) and fully sterilizable (AIM 2) device. SQZ also proposes to expand the application areas to delivery into novel cell types (AIM 3), to enable live cell imaging (AIM 4), to facilitate gene integration into cell lines (AIM 5) and perform a functional analysis (AIM 6). The updated device format will enable a wide range of cell-based assays for both traditional drug discovery applications and research into unchartered new areas. The next generation of sterilizable CellSqueeze, with significantly enhanced capacity, will achieve high percentage delivery of compounds to iPSCs, T-cells, HeLa, and HEK293 cells and efficient delivery to neurons without major changes in cell functions. It will also enable highly specific (Pearson?s coefficient of correlation >0.8) labeling of proteins in live HeLa cells and create HeLa or HEK293 cell lines integrated with a GFP reporter gene with >99% purity after sorting. We will also demonstrate minimal perturbation of CellSqueeze by transcriptome microarray analysis benchmarked against electroporation. At the successful conclusion of this Phase II SBIR, we will be poised to license CellSqueeze to pharmaceutical companies to enable novel and efficient drug screening processes. The commercialization of CellSqueeze will potentially reduce the time and cost of drug discovery and bring efficacious treatments to patients faster.